Immunoglobulins derived from human plasma are used in the treatment of a number of clinical conditions such as primary agammaglobulinaemia, ITP and Kawasaki Syndrome. There is a growing demand for the product as an increasing number of studies are finding that it has application in many other autoimmune conditions. At present, immunoglobulins are purified from plasma by the Cohn fractionation procedure which separates plasma proteins by differential precipitation with ethanol. (Cohn et al., 1946).
The Cohn fractionation process has a number of disadvantages:
1. it is not amenable to automation; PA1 2. it utilises a potentially hazardous chemical, ethanol, which impacts on plant design; PA1 3. it exposes the immunoglobulins to harsh conditions which may affect the function of antibodies; and PA1 4. the yield of immunoglobulins is relatively low (.about.40%). PA1 a. Cohn fractionation is used in most of these processes to separate albumin from immunoglobulins. This prevents the development of an integrated completely chromatographic process. The precipitation of the immunoglobulin fraction followed by resuspension would also result in greater loss of IgG when compared to a process which avoids the need to have a preliminary immunoglobulin enrichment step. PA1 b. The use of cation-exchange chromatography involving conditions which promote binding of immunoglobulins would limit the through-put of the process. It would also be expected that IgG recovery would be reduced and there could be selective depletion of immunoglobulin sub-classes which could limit the clinical acceptance of the product. PA1 c. Where anion-exchange chromatography has been used, conditions of pH have been chosen which have led to sub-class depletion. PA1 d. In none of the processes is there recognition that the degree of loading of the resin can affect the IgM content of the recovered product--an important parameter in defining the clinical usefulness of the final product. PA1 e. Processes exist where plasma has been used directly as the starting material for fractionation, however this is undesirable, as lipoprotein content can promote column fouling. PA1 f. None of the processes have recognised the advantage of macro-porous resins for the production of purified immunoglobulins. PA1 subjecting the plasma or other immunoglobulin-containing material to chromatographic fractionation on a macro-porous anion-exchange resin to recover an immunoglobulin-containing fraction therefrom. PA1 fractionation of the delipidated material by anion-exchange chromatography to produce a first immunoglobulin-containing fraction; and PA1 purification of the first immunoglobulin-containing fraction by a second anion-exchange chromatographic step using a macro-porous anion-exchange resin.
Therefore, there is a need for an efficient process for the recovery of immunoglobulins (IgG) at large scale.
A number of chromatographic processes for the production of pure IgG for intravenous administration have been described. Initially, ion-exchange resins such as DEAE-Sephadex were employed largely for "clean-up" purposes such as the removal of aggregates from Cohn fractionation derived product (Bjorling, 1972; Hoppe et al. 1973). However, integrated chromatographic processes for the recovery of IgG from plasma were subsequently described. Condie described a chromatographic process yielding IgG which was safe for injection (Condie, 1979; Condie 1980). One of the major issues that needed to be addressed was the potential fouling of resins by lipoproteins present in plasma. In this process, fumed colloidal silica (Aerosil) was used to remove lipoprotein from plasma prior to the chromatographic fractionation. However this treatment resulted in a marked decrease of IgG3 subclass under the chosen conditions. This altered subclass distribution is clinically undesirable in an immunoglobulin preparation. Further, removal of the silica was effected by centrifugation which is not amenable to the processing of large batches of immunoglobulin source material. The subsequent ion-exchange chromatography step was carried out using a QAE-Sephadex column at pH 7.0 with imidazole-acetate buffer at an ionic strength of 6.15 mS. Under these conditions, it would be anticipated that there would be increased interaction between immunoglobulin and the resin leading to reduced recovery. In particular, there would be losses of IgG3 and IgG4 which have a relatively anodal charge relative to IgG1 and IgG2. In addition, the use of imidazole-containing buffers or other buffers required to perform the process at pH 7 would significantly add to costs.
Bjorling has presented a procedure for isolation of gamma globulin from defibrinated serum by ion-exchange chromatography on CM-Sepharose CL6B followed by DEAE-Sepharose CL6B (Bjorling, 1985). However, in both these cases the anion-exchange step is carried out at or near pH 7.0, which would be undesirable, as described above. The use of a cation-exchange step under conditions where IgG is bound would require the use of more cycles than if anion-exchange resin was used under conditions where only contaminating proteins are bound. The binding of IgG with the use of cation-exchange chromatography would also result in greater loss of material than with the use of anion-exchange chromatography under conditions where contaminating proteins are retained and immunglobulins are unbound.
Anion-exchange chromatography has also been performed on DEAE-Trisacryl at pH 8.4 (Tousch et al 1989). Under these conditions it would not be applicable to use plasma or plasma fractions such as Cohn Supernatant 1 (SNI) as all proteins would bind thus limiting the effective capacity and through-put. Therefore partial purification by Cohn fractionation was initially performed to remove albumin. Under these chromatographic conditions there would be binding of IgG to the resin. This would most likely result in reduced recovery of protein and compromised sub-class distribution.
Friesen and co-workers described a process for the purification of immunoglobulin (IVIG) using anion exchange at lower pH values. Thus, plasma cryoprecipitate was applied onto DEAE-Sepharose CL6B at pH 5.2, followed by DEAE-Biogel at pH 6.5 or DEAE-Sephadex A-50 at pH 7.5. (Friesen et al. 1985; Friesen, 1982). The DEAE-Sepharose and DEAE-Biogel steps were however carried out in 70 mM and 20 mM acetate respectively, and at pH 5.2 and 6.0. In the light of data presented herein, these buffers do not represent optimal conditions for chromatographic purification of immunoglobulin.
Berglof and co-workers later incorporated Fast Flow Sepharose supports in their process, using the sequence DEAE-Sepharose-FF, Q-Sepharose FF, CM-Sepharose FF (Berglof and Eriksson 1989). This process has lwow capacity capabilities reflecting the less than optimal configuration of the process.
Another process involving ion-exchange chromatography for purifying an immune serum globulin fraction from crude plasma (Samo, 1991) employs cation exchange on CM-Sepharose FF at pH 5-6, followed by anion exchange on DEAE-Sepharose FF at pH 7.0-8.5. The process however requires an initial PEG+solvent/detergent clean-up step of the Cohn fraction I+II+III starting material. Furthermore, it would be expected that the high pH in the anion-exchange step would result in losses of IgG through adsorption to the resin.
The processes described above therefore incorporate many undesirable features. These include the following:
In the work leading to the present invention, it has been found that chromatographic processes for the purification of immunoglobulins from plasma and other immunoglobulin-containing materials can be enhanced by use of a macro-porous anion-exchange resin. In addition, where the starting material contains lipoproteins, the purification processes can also be enhanced by introducing a pretreatment step wherein lipoproteins are removed by adsorption prior to application of the starting material onto the chromatography column.